RNA extraction, real-time PCR, and the Human Epithelial to Mesenchymal Transition (EMT) RT2 Profiler PCR Array

Total RNA from NPC specimens and cultured cells was extracted and real-time PCR was performed as previously described [14]. Reverse transcription was performed using reverse transcriptase (Promega, Madison, WI, USA) with random primers (Promega) for genes or Bulge-Loop miRNA-specific RT-primers (RiboBio, Guangzhou, China) for miR-142-3p. Real-time PCR reactions were performed with SYBR Green qPCR SuperMix-UDG reagents (Invitrogen) on a CFX96 Touch sequence detection system (Bio-Rad, Hercules, CA, USA). GAPDH and U6 were used as internal controls for genes and miR-142-3p, respectively. The Human Epithelial to Mesenchymal Transition (EMT) RT² Profiler PCR Array (Qiagen, Hilden, Germany) was performed on a LightCycler 480 system (Roche, Basel, Switzerland), according to the manufacturer’s instructions. The relative expression was calculated with the 2^−ΔΔCT equation [52].